Method for producing recombinant virus

ABSTRACT

The invention concerns a method for producing recombinant virus. This method is based on the use of baculovirus for providing the complementary functions. It also concerns constructs used for implementing this method, the producing cells, and the resulting virus.

[0001] The present invention relates to a method for the production ofrecombinant viruses. It also relates to constructs used for carrying outthis method, the producing cells, and the viruses thus produced. Theseviruses can be used as vector for the cloning and/or expression of genesin vitro, ex vivo or in vivo.

[0002] Vectors of viral origin are widely used for the cloning, transferand expression of genes in vitro (for the production of recombinantproteins, for carrying out screening tests, for studying the regulationof genes and the like), ex vivo or in vivo (for the creation of animalmodels, or in therapeutic approaches). Among these viruses, there may bementioned in particular adenoviruses, adeno-associated viruses (AAV),retroviruses, herpesviruses or vaccinia viruses.

[0003] The Adenoviridae family is widely distributed in mammals andbirds and comprises more than one hundred different serotypes ofnonenveloped double-stranded DNA viruses possessing a capsid oficosahedral symmetry (Horwitz, In: Fields B N, Knipe D M, Howley P M,ed. Virology. Third edition ed. Philadelphia: Raven Publishers, 1996:2149-2171). In addition to its safety, the adenovirus has a very broadcellular tropism. Unlike the retrovirus, whose cycle is dependent oncell division, it can infect actively dividing cells such as quiescentcells and its genome is maintained in episomal form. Furthermore, it canbe produced at high titres (10¹¹ pfu/ml). These major assets of it onemakes a most preferred vector for the cloning and expression ofheterologous genes. The group C adenoviruses, particularly types 2 and5, as well as the CAV-2-type canine adenoviruses, whose molecularbiology is best known, are the source of the vectors currently used.

[0004] The adenovirus has a linear genome of 36 kb, terminating at eachof these ends with inverted terminal repeat (ITR) sequences of 103 bpcomprising a replication origin as well as an encapsidation signalsituated near the left ITR (Shenk, Adenoviridae: The Viruses and TheirReplication. In: Fields B N, Knipe D M, Howley P M, ed. Virology.Philadelphia: Raven publishers, 1996: 2111-2148). Three families ofgenes are expressed during the viral cycle:

[0005] The immediate-early genes (E1, E2, E3 and E4) which are involvedin the regulation of cellular genes allowing in particular the entry ofthe cell into the S phase (E1A) and the inhibition of apoptosis (E1B).They are also involved in the regulation of early or late viral genes atthe level of the transcription, splicing or transport of the messengerRNAs (E1A E2A, E4). They also play a role in replication and in escapingthe immune response.

[0006] The delayed-early genes (pIX and IVa2) are linked to theregulation of transcription of the late genes (IVa2) or to theassembling of the virion (pIX).

[0007] The late genes (L1 to L5) are transcribed from the strongpromoter (MLP). A primary transcript of 28 kb makes it possible togenerate the transcripts corresponding to the various structuralproteins (core, penton, hexon) and nonstructural proteins participatingin the assembling and in the maturation of the viral particles, byalternative splicing and the use of 5 polyadenylation sites.

[0008] Adenoviral vectors have been used for the cloning and expressionof genes in vitro (Gluzman et al., Cold Spring Harbor, N.Y. 11724, p.187), for the creation of transgenic animals (WO95/22616), for thetransfer of genes into cells ex vivo (WO95/14785; WO95/06120) or for thetransfer of genes into cells in vivo (see in particular WO93/19191,WO94/24297, WO94/08026).

[0009] As regards the adeno-associated viruses (AAV), they arerelatively small DNA viruses which become integrated into the genome ofthe cells which they infect, in a stable and relatively site-specificmanner. They are capable of infecting a broad spectrum of cells, withoutinducing any effect on cell growth, morphology or differentiation.Moreover, they do not seem to be involved in pathologies in man. Thegenome of the AAVs has been cloned, sequenced and characterized. Itcomprises about 4700 bases and contains, at each end, an invertedterminal repeat (ITR) region of about 145 bases which serves asreplication origin for the virus. The remainder of the genome is dividedinto 2 essential regions carrying the encapsidation functions: the leftpart of the genome, which contains the rep gene involved in the viralreplication and the expression of the viral genes; the right part of thegenome, which contains the cap gene encoding the virus capsid proteins.

[0010] The use of vectors derived from AAVs for the transfer of genes invitro and in vivo has been described in the literature (see inparticular WO91/18088; WO93/09239; U.S. Pat. No. 4,797,368, 5,139,941,EP 488 528).

[0011] As regards the retroviruses, they are integrative viruses whichselectively infect dividing cells. They therefore constitute vectors ofinterest for cancer or restenosis applications for example. The genomeof retroviruses essentially comprises two LTRs, an encapsidationsequence and three coding regions (gag, pol and env). The constructionof recombinant vectors and their use in vitro or in vivo has been widelydescribed in the literature: see in particular Breakfield et al., NewBiologist 3 (1991) 203; EP 453242, EP 178220, Bernstein et al. Genet.Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like.

[0012] For their use as recombinant vectors, various constructs derivedfrom viruses have been prepared, incorporating various genes ofinterest. In each of these constructs, the viral genome was modified soas to make the virus incapable of autonomously replicating in theinfected cell. Thus, the constructs described in the prior art areviruses which are defective for certain regions of their genome whichare essential for replication. In particular, as regards adenoviruses,the first-generation constructs exhibit a deletion in/of the E1 region,which is essential for viral replication, at the level of which theheterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991)195; Gosh-Choudhury et al., Gene 50 (1986) 161). Moreover, to enhancethe properties of the vector, it has been proposed to create otherdeletions or modifications in the adenovirus genome. Thus, aheat-sensitive point mutation was introduced into the ts125 mutant,making it possible to inactivate the 72 kDa DNA binding protein (DBP)encoded by the E2 region (Van der Vliet et al., 1975). Other vectorscomprise a deletion of another region essential for the viralreplication and/or propagation, the E4 region. Adenoviral vectors inwhich the E1 and E4 regions are deleted have highly reducedtranscription background noise and viral gene expression. Such vectorshave been described, for example, in applications WO94/28152,WO95/02697, WO96/22378. Moreover, vectors carrying a modification at thelevel of the IVa2 gene have also been described (WO96/10088). Inaddition, so-called “minimum adenovirus” or “pseudo-adenovirus” vectors(or alternatively AdΔ) containing only the regions necessary in cis forthe production of the virus (ITR and encapsidation sequences) andlacking any coding viral sequence have also been described (WO94/12649,WO94/28152, WO95/02697), although their production remains verydifficult, as explained below.

[0013] As regards the AAVs, the vectors described generally lack theentire coding regions Rep and Cap, which are replaced by nucleic acidsof interest.

[0014] In the recombinant vectors derived from retroviruses, the gag,pol and env genes are generally deleted, completely or in part, andreplaced by a heterologous nucleic acid sequence of interest. Moreover,the recombinant retroviruses may comprise modifications at the level ofthe LTRs in order to suppress the transcriptional activity, as well aslarge encapsidation sequences, comprising part of the gag gene (Benderet al., J. Virol. 61 (1987) 1639).

[0015] Given their defective character in relation to the replication,the production of these various recombinant viruses involves thepossibility of transcomplementing the functions deleted from the genome.The transcomplementation is precisely the source of major difficultiesfor the production of these viruses, and in particular the provision ofthe transcomplementation functions.

[0016] Two approaches have been developed in this regard. The first isbased on the construction of transcomplementing lines, that is to sayencapsidation lines. The second is based on the use of helperadenoviruses or of helper plasmids.

[0017] Various encapsidation lines of defective viruses have beenconstructed. These lines are capable of producing the functionsdeficient in the viral vector. Generally, these lines comprise,integrated into their genome, the region(s) deleted from the viralgenome (E1, E2 and/or E4 for example for the adenovirus; gag, pol and/orenv for the retrovirus, rep and/or cap for the AAV).

[0018] One of the lines known for the production of defectiveadenoviruses is for example the line 293 into which part of theadenovirus genome has-been integrated. More precisely, the line 293 is ahuman embryonic kidney cell line containing the left end (about 11-12%)of the serotype 5 adenovirus (Ad5) genome, comprising the left ITR, theencapsidation region, the E1 region, including E1a and E1b, the regionencoding the protein pIX and part of the region encoding the proteinpIVa2. This line is capable of transcomplementing recombinantadenoviruses defective for the E1 region, that is to say lacking all orpart of the E1 region, and of producing viral stocks having high titres.This line is also capable of producing, at permissive temperature (32°C.), virus stocks comprising, in addition, the heat-sensitive E2mutation. Other cell lines capable of complementing the E1 region havebeen described, based in particular on human lung carcinoma cells A549(WO94/28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215).Moreover, lines capable of transcomplementing several functions in theadenovirus have also been described. In particular, there may bementioned lines complementing the E1 and E4 regions (Yeh et al., J.Virol. 70 (1996) 559; Cancer Gen. Ther. 2 (1995) 322; Krougliak et al.,Hum. Gen. Ther. 6 (1995) 1575) and lines complementing the E1 and E2regions (WO94/28152, WO95/02697, WO95/27071).

[0019] Various lines have also been described for the production ofdefective retroviruses, generally capable of expressing the gag, pol andenv genes. Such lines are, for example, the PA317 line (U.S. Pat. No.4,861,719), the PsiCRIP line (WO90/02806), the GP+envAm-12 line(WO89/07150), the BOSC line (WO94/19478) and the like. To constructrecombinant retroviruses comprising a nucleic acid of interest, aplasmid comprising in particular the LTRs, the encapsidation sequenceand the said nucleic acid is constructed, and then used to transfect anencapsidation line as described above, capable of providing in trans theretroviral functions deficient in the plasmid. The recombinantretroviruses produced are then purified by conventional techniques.

[0020] The use of lines can however have certain disadvantages. Thus, itis difficult, expensive and restrictive at the industrial level toconstruct and to validate such lines. Indeed, these lines should bestable and compatible with industrial uses. Furthermore, the linesdescribed hardly make it possible to avoid the production of replicativecontaminant viruses (RCA). Moreover, these lines do not allow at thepresent time, in a satisfactory manner for an industrial use, veryhighly defective viral genomes, such as for example minimum adenovirusesas described above, to be transcomplemented. Indeed, the adenovirus hasa genome organized into various transcription units whose spatiotemporalregulation is very complex. It has so far not been possible to carry outsatisfactorily the transcomplementation of an adenovirus deleted of allthe coding viral sequences by expressing each transcription unitseparately, in a constitutive or conditional manner, using a cell line.Thus, only a small proportion of the genome corresponding to the E1, E4and pIX regions, and to the three proteins encoded by E2 (pol, DBP andp-TP) has been constitutively expressed using cell lines. The remainderof the genome corresponds to the major late transcription unit (MLTU)which produces all the messengers for the structural and nonstructuralproteins from a primary transcript of 28 kb and is activated afterreplication of the genome. Now, to generate minimum adenoviruses,transcomplementation of these regions is essential. Neither do theselines make it possible to obtain very high recombinant retrovirustitres.

[0021] The second approach consists in cotransfecting with the defectiveviral genome a construct (plasmid or adenovirus), providing thecomplementation functions. In particular, the defective recombinant AAVsare generally prepared by cotransfection, in a cell line infected by ahuman helper virus (for example an adenovirus), of a plasmid containinga nucleic sequence of interest bordered by two AAV inverted terminalrepeat (ITR) regions, and of a plasmid carrying the AAV complementationfunctions (rep and cap genes). Variants have been described inapplications WO95/14771; WO95/13365; WO95/13392 or WO95/06743. Thedisadvantage of using a helper adenovirus lies mainly in the increasedrisks of recombination between the adenoviral vector and the helperadenovirus, and in the difficulty of separating the recombinant from thehelper during the production and purification of the viral stocks. Thedisadvantage of using a helper plasmid, for example a plasmid Rep/cap,lies in the transfection levels obtained, which do not make it possibleto produce high virus titres.

[0022] The present application now describes a new system for theproduction of viruses which makes it possible to overcome thesedisadvantages. The system of the invention is based on the use of abaculovirus to provide the complementation functions.

[0023] The production system according to the invention makes itpossible, in a particularly advantageous manner, to dispense with theuse of established complementation lines, to avoid the problems of RCA,and to transcomplement highly defective genomes. In addition, the systemof the invention is applicable to any cell capable of being infected bythe desired virus and by a baculovirus, and thus offers greatflexibility of use.

[0024] A first subject of the invention therefore consists in a processfor the production of defective recombinant viruses according to whichthe genome of the defective recombinant virus and a baculoviruscomprising all or some of the functions necessary for thetranscomplementation of the defective recombinant genome are introducedinto a population of competent cells.

[0025] The process of the invention is therefore based on the use of abaculovirus to provide the complementing functions. Various approachesare possible. It is possible, first of all, to use competent cells notexpressing any function of transcomplementation of the defectiverecombinant genome. In this case, it is possible to use either abaculovirus comprising all the functions necessary for thetranscomplementation of the defective recombinant genome, or severalbaculoviruses each carrying one or more of the functions necessary forthe transcomplementation of the defective recombinant genome. It is alsopossible to use a population of competent cells capable of alreadytranscomplementing one or more functions of the defective recombinantgenome (encapsidation line). In this case, the baculovirus(es) used willprovide only the functions necessary for the transcomplementation of thedefective recombinant genome which are not already transcomplemented bythe competent cells.

[0026] As indicated above, the advantages of the system of the inventionare numerous in terms of industrialization (no need for lines, no RCA,and the like), and in terms of applications (production of recombinantviruses carrying any type of deletion, and particularly of highlydefective recombinant adenoviruses). In addition, since the baculovirusdoes not replicate in human cells, the viral preparation obtained is notcontaminated by the baculovirus. Furthermore, the baculovirus beingphylogenetically very distant from adenoviruses, there is no risk ofrecombination or transcomplementation between the two. This systemtherefore makes it possible, in an advantageous manner, to produceconcentrated stocks of defective viruses, lacking RCA. This system ismost particularly advantageous for the production of defectiverecombinant adenoviruses.

[0027] Baculoviruses are enveloped, circular double-stranded DNA virusesspecific for invertebrates. Their prototype, AcNPV, has a genome of 133kb. It is widely used as vector for the expression of eukaryotic genesin insect cells, starting from two strong promoters [polyhedrin (Ph) andP10], (King and Possee, The baculovirus expression system. London:Chapman & Hall, 1992.) AcNPV is capable of infecting some mammaliancells, but the genome is neither transcribed nor translated. Recently,Hofmann et al. (PNAS 92 (1995) 10099) have shown that in vitro,hepatocytic cells can be transduced by a purified recombinantbaculovirus expressing the LacZ gene. No cellular toxicity was reported,even with a multiplicity of infection of 1000, and the transfectionefficiency described in this article is about 50% for an MOI of 100.

[0028] The applicant has now shown that it is possible to infect variouscell types with a recombinant baculovirus. In particular, the applicanthas shown that it was possible, with a recombinant baculovirus, toinfect cells of human origin such as immortalized embryonic cells. Theapplicant has also shown that it is possible to obtain a very hightransduction efficiency (>80%). The applicant has also shown that it ispossible to introduce, into a baculovirus, functions for complementationof an adenovirus, and to express these functions in a population ofcompetent cells. The applicant has thus made it possible to show thatthe baculovirus constitutes an inert vector which can be advantageouslyused for the transfer and expression of virus complementation functionsinto mammalian, particularly human, cells. Other advantages of thesystem of the invention are in particular (i) the large cloning capacitywhich makes it possible to complement a whole adenoviral genome and (ii)the advanced development of the technology of the baculovirus.

[0029] The baculovirus carrying the functions for complementation of thevirus is also designated in the text which follows helper baculovirus.It may comprise various functions for complementation of the virus.

[0030] Thus, the helper baculovirus may comprise the E1 region of theadenovirus. A Baculo-E1 can be used for the production offirst-generation adenoviruses, that is to say adenoviruses defective forthe E1 region (AdΔE1), regardless of its E3 status (i.e. defectiveAdΔE1, ΔE3, or not). The production of first-generation defectiverecombinant adenoviruses (defective for the E1, and possibly E3, region)constitutes a first particularly advantageous application of the processof the invention. As indicated above, various lines have been describedin the literature which are capable of transcomplementing the E1function (cells 293, cells A549, cells 911, and the like). However,various zones of homology exist between the region carrying thetranscomplementation functions which is integrated into the genome ofthe line and the DNA of the recombinant virus which it is desired toproduce. Because of this, during production, various recombinationevents may occur, generating replicative viral particles, in particulartype E1+adenoviruses. This may be a single recombination event followedby breaking of the chromosome, or a double recombination. These twotypes of modification lead to reintegrating into its initial locuswithin the adenoviral genome the E1 region contained in the cellulargenome. Moreover, given the high titres of recombinant vector which areproduced by the line 293 (greater than 10¹²), the probability of theserecombination events occurring is high. In fact, it has been observedthat numerous batches of first-generation defective recombinantadenoviral vectors were contaminated by replicative viral particles,which may constitute a major disadvantage for pharmaceutical uses.Indeed, the presence of such particles in therapeutic compositions wouldinduce in vivo an uncontrolled viral propagation and dissemination withrisks of inflammatory reaction, of recombination and the like. Thecontaminated batches cannot therefore be used in human therapy.

[0031] The present invention makes it possible to overcome thesedisadvantages. Indeed, according to one embodiment of the process of theinvention, the genome of the recombinant adenovirus defective for theE1, and possibly E3, region is introduced into the competent cells,these cells are infected, simultaneously or otherwise, with abaculovirus comprising the E1 region, the adenovirus E1 region presentin the baculovirus and the genome of the defective recombinantadenovirus comprising no zone of homology (overlapping) capable ofgiving rise to recombination. According to this embodiment, it is thuspossible to rapidly produce, without an established line, stocks offirst-generation recombinant adenoviruses free of RCA. Moreover, asindicated below, the stocks of recombinant adenoviruses thus generated,free of RCA, can be used as starting material for a new production, bycoinfection in the competent cells with a baculovirus.

[0032] The helper baculovirus may also comprise the E2 region of theadenovirus, in full or in part, particularly the E2a and/or E2b region.A baculo-E2 may be used to produce, in competent cells, adenovirusesdefective for the E2 region (Ad-ΔE2), and possibly for the E3 region(Ad-ΔE2, ΔE3). In addition, in competent cells capable of complementingthe E1 region of the adenovirus, the baculo-E2 may allow the productionof recombinant adenoviruses defective for the E1 and E2 (Ad-ΔE1, ΔE2)and possibly E3 (Ad-ΔE1, ΔE2, ΔE3) regions. Likewise, in competent cellscapable of complementing the E1 and E4 regions of the adenovirus (forexample in IGRP2 cells), the baculo-E2 may allow the production ofrecombinant adenoviruses defective for the E1, E2 and E4 (Ad-ΔE1, ΔE2,ΔE4) and possibly E3 (Ad-ΔE1, ΔE2, ΔE3, ΔE4) regions.

[0033] The helper baculovirus may also comprise the E4 region (in fullor in part) of the adenovirus. A baculo-E4 may be used to produce, incompetent cells, adenoviruses defective for the E4 region (Ad-ΔE4), andpossibly for the E3 region (Ad-ΔE4, ΔE3). In addition, in competentcells capable of complementing the E1 region of the adenovirus, thebaculo-E4 may allow the production of recombinant adenoviruses defectivefor the E1 and E4 (Ad-ΔE1, ΔE4) and possibly E3 (Ad-ΔE1, ΔE4, ΔE3)regions.

[0034] The helper baculovirus may also comprise the E1 and E4 regions(in full or in part) of the adenovirus. A baculo-E1, E4 may be used toproduce, in competent cells, adenoviruses defective for the E1 and E4(Ad-ΔE1, ΔE4) and possibly E3 (Ad-ΔE1, ΔE4, ΔE3) regions, as illustratedin FIG. 1.

[0035] In addition, to generate viruses defective for the E1 and E4regions, it is also possible to use two helper baculoviruses, oneexpressing the E1 function, the other the E4 function, in full or inpart.

[0036] In the same manner, the helper baculovirus may comprise the E1,E2 and E4 regions (in full or in part), and possibly the regionscarrying the late genes (L1-L5).

[0037] The helper baculovirus may also comprise the AAV Rep and/or Capregions. A baculo-Rep/Cap thus makes it possible to complement, in aline of competent cells, an AAV genome lacking any coding viral sequence(FIG. 5).

[0038] The baculovirus may also comprise the gag, pol and/or env regionsof a retrovirus. A baculo-gag/pol/env thus makes it possible tocomplement, in a line of competent cells, a retroviral genome lackingany coding viral sequence.

[0039] It is also possible to use a baculovirus comprising the gag/polregions and a second baculovirus containing the env region.

[0040] In general, it is preferable that the genome of the defectiverecombinant virus and the complementation regions present in thebaculovirus do not overlap. This makes it possible, indeed, to avoid therisks of recombination and thus the generation of RCA. This isparticularly important for the generation of first-generationadenoviruses (Ad-ΔE1). In this case, the E1 region introduced into thebaculovirus is defined so that it does not possess any common sequencewith the recombinant genome. To do this, it is possible, for example, todelete from the recombinant genome a region larger than thecomplementing region inserted into the baculovirus, as illustrated inthe examples. This is also advantageous for the generation of Ad-ΔE1,ΔE4 adenovirus.

[0041] Thus, in a specific embodiment of the process of the invention,the genome of the defective recombinant virus is introduced into thecompetent cells, these cells are infected, simultaneously or otherwise,with a baculovirus comprising all or some of the functions necessary forthe complementation of the defective genome, the complementationfunctions present in the baculovirus and the genome of the defectiverecombinant virus comprising no zone of homology capable of giving riseto recombination. Advantageously, the viral genome is a recombinantadenovirus genome defective for the E1 region and the baculoviruscarries a region of the adenovirus capable of transcomplementing the E1region. According to another variant, the viral genome is a recombinantadenovirus genome defective for the E1 and E4 regions and thebaculovirus carries two adenovirus regions capable of transcomplementingthe said regions or two baculoviruses are used, one carrying a region ofthe adenovirus capable of transcomplementing the E1 region and the othera region of the adenovirus capable of transcomplementing the E4 region,without zones of homology with the defective adenoviral genome.

[0042] According to a specific embodiment, all the coding regions of theadenovirus are carried by one or more helper baculoviruses. According toa more specific embodiment, only one helper baculovirus comprising allthe coding regions of the adenovirus is used. Such a helper baculoviruscan thus be used to transcomplement minimum recombinant adenoviruses.Such a baculovirus may in particular comprise the whole of oneadenoviral genome, with the exception of the encapsidation region andpossibly the ITRS, as illustrated in the examples.

Preparation of the Complementation Functions

[0043] The complementation functions introduced into the helperbaculovirus may be derived from viruses of different serotypes.

[0044] As regards adenoviruses, various serotypes exist whose structureand properties vary somewhat, but which exhibit a comparable geneticorganization. More particularly, the complementation functions used forthe construction of the baculoviruses according to the invention arederived from an adenovirus of human or animal origin.

[0045] As regards adenoviruses of human origin, there may be mentioned,preferably, those classified in the C group. Still more preferably,among the various human adenovirus serotypes, the use of the type 2 or 5adenoviruses (Ad2 or Ad5) is preferred within the framework of thepresent invention. It is also possible to use regions derived from type7 or 12 adenoviruses, belonging to groups A and B. Among the variousadenoviruses of animal origin, the use of the adenoviruses of canineorigin, and particularly all the strains of the CAV2 adenoviruses[Manhattan or A26/61 strain (ATCC VR-800) for example] is preferredwithin the framework of the invention. Other adenoviruses of animalorigin are mentioned in particular in application WO94/26914incorporated into the present by reference.

[0046] In a preferred embodiment of the invention, the complementationfunction is derived from a group C human adenovirus genome. Morepreferably, it is derived from the genome of an Ad2 or Ad5 adenovirus.

[0047] The regions carrying the various complementation functions may beobtained, from an adenoviral genome, by enzymatic cleavages according tomethods known to persons skilled in the art. These regions mayoptionally be modified in order to reduce their size, or to replacecertain regulatory elements (promoter, enhancer and the like) withheterologous elements. In general, these regions are prepared asfollows: the DNA of an adenovirus is purified by caesium chloridegradient centrifugation or obtained in vitro from a prokaryotic(WO96/25506) or eukaryotic (WO95/03400) plasmid. The DNA is then cleavedwith appropriate restriction enzymes and the fragments obtained,carrying the desired complementation functions, are identified andselected. The choice of the restriction enzymes used depends on thedesired complementation functions. It is then guided by the restrictionmaps and the published sequences of the adenoviral genomes. Thus, the E1region may be isolated in the form of fragments carrying all the readingframes of E1A and E1B downstream of the E1A promoter. The E4 region maybe isolated in the form of fragments carrying the whole of the readingframes, or only part of them, and preferably the frames ORF3 or ORF6 orORF6-ORF6/7.

[0048] Similar methodologies are used to prepare the AAV and recombinantretrovirus complementation regions. Thus, the AAV rep and/or cap regionsmay be obtained by enzymatic cleavage from the viral DNA isolated fromvarious AAV serotypes. This is preferably AAV-2. For retroviruses, thegag, pol and/or env regions may also be obtained according toconventional molecular biological techniques, from various types ofretroviruses, such as in particular MoMuLV (Murine Moloney LeukaemiaVirus; also called MoMLV), MSV (Murine Moloney Sarcoma Virus), HaSV(Harvey Sarcoma virus); SNV (Spleen Necrosis Virus), RSV (Rous SarcomaVirus) or Friend's virus.

Construction of the Helper Baculovirus

[0049] The fragments carrying the complementation regions are thensubcloned into a plasmid vector allowing their manipulation (finerdigestions, PCR, additions of regulatory sequences, and the like), forexample in a prokaryotic or eukaryotic organism. The final fragmentobtained, encoding the complementation function(s) is then introducedinto the helper baculovirus using conventional molecular biologicaltechniques. Specifically, the fragment is cloned between two sequenceshomologous to a region of the genome of a baculovirus, and then theresulting fragment or plasmid is cotransfected with the genome of abaculovirus into insect cells (conventionally Sf9 and Sf21, spodopterafrugiperda cells, but also Tn-368 and High-Five™ BTI-TN-5B1-4 (Gibco),trichopulsia ni cells, or any other insect cell permissive tobaculoviruses and capable of being used for their production). Thehomologous recombination between the plasmid or fragment and the genomeof the baculovirus generates the desired recombinant baculovirus, whichmay be recovered and purified according to conventional methods (see inparticular King and Possee: the baculovirus expression system. London:Chapman & Hall, 1992). For the construction of the recombinantbaculoviruses, various kits comprising shuttle vectors arecommercialized and may be used according to the recommendations of themanufacturers. In particular, it is possible to use the shuttle vectorspBAC marketed by the company Clontech, the vectors pAc (Verne et al.,Bio/Technology 6 (1988) 47, Pharmingen, USA), the vectors pBlue-Bac(Invitrogen) or the vectors pBSV (Boehringer). The complementationfunctions may thus be inserted into different sites of the baculovirus,and in particular into the locus of the polyhedrin gene or of the p10gene. Moreover, various baculovirus strains can be used, such as inparticular AcNPV or Bombyx mori (Maeda et al., Nature 315 (1988) 592).Furthermore, the baculovirus used may be modified to enhance/change itstropism. It is indeed possible to modulate the tropism of the viralvectors by modifying their surface proteins so as (i) to limit it byfusion of the viral proteins with a specific ligand (lightimmunoglobulin chain, Gastrin-Releasing Peptide) or (ii) to broaden itby formation of pseudotypes with a heterologous viral glycoprotein [G ofthe Vesicular Stomatitis Virus (VSV)], [Liu et al., J. Virol 70(4)(1996) 2497; Michael et al., Gene Ther. 2 (1995) 660]. Recently, it wasshown that the baculovirus surface glycoprotein (gp64) fused with gp120of the HIV virus was capable of binding to the CD4 receptor (Boublik etal. Bio/Technology 13 (1995) 1079). This modification of gp64 does notaffect the viability of the baculovirus in insect cells. A similarconstruct with the G of VSV should make it possible to enhance thetropism of the baculovirus for mammalian cells and therefore to increasethe transduction efficiency of the Huh7 cells as well as other celllines.

[0050] In the helper baculovirus, the complementation functions areadvantageously placed under the control of a heterologous promoter (i.e.of a different origin from the baculovirus), which is functional incompetent cells. It appears, indeed, that the baculovirus promoters donot make it possible to obtain sufficient levels of expression of thecomplementation functions in cells other than insect cells, and aretherefore not the most appropriate for the applications of theinvention. The promoter may first of all be the actual promoter(homologous promoter) of the complementation functions of the virus(E1A, E4, E2, MLP promoter for the adenovirus, P5 or P19 promoters ofAAV, the LTR promoter of RSV, and the like). It may also be any promoterof different origin which is functional in the competent cell used. Tothis effect, there may be mentioned for example the promoters of genesexpressed in this cell, or known ubiquitous promoters such as forexample the promoter of the PGK gene, the immediate-early promoter ofCMV, the promoter of the TK gene of the herpesvirus or alternatively theLTR promoter of RSV. It may also be a regulated promoter, such as forexample the promoter of the MMTV virus, a promoter responding tohormones, for example of the GRE5 type, or a promoter regulated bytetracycline (WO). Advantageously, it is an inducible or strongubiquitous, homologous promoter.

[0051] Thus, another subject of the present invention relates to arecombinant baculovirus comprising, inserted into its genome, a nucleicacid encoding a complementation function of a virus placed under thecontrol of a heterologous promoter. More particularly, thecomplementation function is a protein necessary for the production ofthe said virus, and whose coding region is inactive (mutated, deletedand the like) in the defective viral genome. For adenoviruses, thecomplementation function is more particularly chosen from all or some ofthe functions encoded by the E1, E2, E4, L1-L5, pIX and IVa2 regions ofthe adenovirus, alone or in combination. For the AAV, they are functionsencoded by the Rep and/or Cap regions; and for the retrovirus, gag, poland/or env. Advantageously, the nucleic acid corresponds to a region ofa viral genome comprising the region encoding the complementationfunction chosen. In particular, it is a fragment of a genome ofadenoviruses of serotype Ad2 or Ad5, MOMLV or AAV-2. In a particularlypreferred manner, the nucleic acid also comprises the promoter regionwhich is naturally responsible for the expression of the complementationfunctions chosen.

[0052] By way of a specific example, the present invention relates to abaculovirus comprising all or part of the E1 region of an adenovirus.More particularly, it is a baculovirus comprising the E1a, E1b or E1aand E1b region. The E1 region of the adenovirus is located at the levelof nucleotides 104 (promoter E1a) to 4070 (polyA E1b) of Ad5. Inparticular, the TATA box of the E1a promoter is located at the level ofnucleotide 470, the ATG codon of E1a at the level of nucleotide 560, andthe stop codon E1b at the level of nucleotide 3511. There may bementioned by way of precise example a baculovirus comprising a fragment391-3511. This helper baculovirus is particularly suitable for theproduction of recombinant adenoviruses defective for the E1 region,carrying a larger deletion than this 391-3511 fragment. In particular,it is suitable for the production of first-generation adenoviruses,without RCA, carrying a deletion in the E1 region covering nucleotides383-3512 inclusive.

[0053] Another specific example of a baculovirus according to theinvention comprises, for example, all or some of the E1 and E4 regionsof the adenovirus. The E4 region of the adenovirus consists of 7 openreading frames, designated ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6 andORF6/7. Among the proteins encoded by these various ORFs, those producedby ORF3 and ORF6 appear to allow the “replication” of the virus, andtherefore the transcomplementation of an adenovirus defective for the E4region, even in its entirety. As a result, the helper baculovirus of theinvention advantageously comprises all the E4 region or only partthereof comprising at least the ORF3 or ORF6 frame. The various parts ofthe E4 region may be obtained by enzymatic cleavages or modifiedaccording to methods known to persons skilled in the art. In particular,the reading frame ORF6 may be isolated from the E4 region in the form ofa BglII-PvuII fragment, corresponding to nucleotides 34115-33126, andthe reading frames ORF6-ORF6/7 may be isolated from the E4 region in theform of a BglII-BglII fragment corresponding to nucleotides 34115-32490of the genome of Ad5. The baculovirus may also comprise the whole of thereading frames ORF1-ORF7 (for example in the form of a 32800-35826 or32811-35614 or 32811-35640 fragment). It is understood that otherfragments may be determined on the basis of published sequences of theadenoviral genomes. The use of a baculovirus carrying a reduced unit ofthe E4 region is advantageous because it allows the transcomplementationof a defective adenoviral genome carrying a larger deletion of the E4region, therefore without a zone of homology, and thus to avoid anypossibility of recombination.

[0054] According to a first embodiment, the nucleic acid encoding thecomplementation function(s) is introduced into the helper baculovirus inthe form of an expression cassette. This embodiment is the easiest touse. It is particularly suitable for the production of recombinantadenoviruses defective for immediate-early genes and for the productionof defective recombinant AAVs and retroviruses.

[0055] According to another embodiment, the nucleic acid encoding thecomplementation function(s) is introduced into the helper baculovirus inthe form of an excisable cassette, generating a replicative molecule inthe competent cell. The replication of the cassette in the cell makes itpossible advantageously to increase the copy number of the complementinggenes, and thus to enhance the production levels of the system. Thisembodiment is particularly suitable for the production of very highlydefective recombinant adenoviruses, particularly defective for thestructural genes. In particular, this embodiment is particularlysuitable for the production of “minimum” adenoviruses. Indeed, thequantity of structural protein is a limiting factor for the productionof high titres of highly defective adenoviruses (minimum adenovirustype). This embodiment makes it possible, for the first time, toconsiderably increase the intracellular levels of transcomplementingproteins, particularly of structural proteins of the adenovirus (encodedby the L1 to L5 regions), up to levels compatible with thetranscomplementation of minimum adenoviruses.

[0056] Thus, the applicant has shown that it is possible to constructrecombinant baculoviruses comprising a heterologous region capable ofbeing excised in a cell, preferably in an inducible and regulatedmanner, in order to generate a circular and replicative molecule (ofepisomal type).

[0057] The excision is advantageously carried out by a site-specificrecombination mechanism, and the replication in the cell is broughtabout by a replication origin, independent of the state of celldivision.

[0058] More preferably, the site-specific recombination used accordingto the process of the invention is obtained by means of two specificsequences which are capable of recombining with each other in thepresence of a specific protein, generally called recombinase. Thesespecific sequences, arranged in the appropriate orientation, flank inthe baculovirus the sequences encoding the complementation functions.Thus, the subject of the invention is also a recombinant baculoviruscomprising, inserted into its genome, at least one DNA region flanked bytwo sequences allowing a site-specific recombination and positioned indirect orientation, the said DNA region comprising at least onereplication origin functional in competent cells and a nucleic acidencoding a complementation function of a virus.

[0059] The sequences allowing the recombination which are used in theframework of the invention generally comprise from 5 to 100 base pairs,and more preferably less than 50 base pairs. They may belong todifferent structural classes, and in particular to the family of therecombinase of the P1 bacteriophage or of the resolvase of a transposon.

[0060] Among the recombinases belonging to the bacteriophage 1 integrasefamily, there may be mentioned in particular the integrase of phageslambda (Landy et al., Science 197 (1977) 1147), P22 and F80 (Leong etal., J. Biol. Chem. 260 (1985) 4468), HP1 of Haemophilus influenzae(Hauser et al., J. Biol. Chem. 267 (1992) 6859), the Cre integrase ofthe P1 phage, the integrase of the plasmid pSAM2 (EP 350 341) or the FLPrecombinase of the plasmid 2 μm of the yeast Saccharomyces cerevisiae.

[0061] Among the recombinases belonging to the Tn3 transposon family,there may be mentioned in particular the resolvase of the Tn3 transposonor of the gd, Tn21 and Tn522 transposons (Stark et al., 1992); the Gininvertase of the mu bacteriophage or the resolvase of plasmids, such asthat of the fragment par of RP4 (Abert et al., Mol. Microbiol. 12 (1994)131).

[0062] According to a preferred embodiment, in the recombinantbaculoviruses of the present invention, the sequences allowing thesite-specific recombination are derived from a bacteriophage. Morepreferably, they are sequences for attachment (attP and attB sequences)of a bacteriophage or of derived sequences. These sequences are capableof specifically recombining with each other in the presence of arecombinase called integrase. By way of specific examples, there may bementioned in particular the sequences for attachment of the phageslambda, P22, F80, P1, HP1 of Haemophilus influenzae or of the plasmidpSAM2, or 2 μm.

[0063] Still more preferably, the sequences allowing the site-specificrecombination are represented by the recombination system of the P1phage. The P1 phage possesses a recombinase called Cre whichspecifically recognizes a nucleotide sequence of 34 base pairs calledlox P site (Sternberg et al., J. Mol. Biol. 150 (1981) 467). Thissequence is composed of two palindromic sequences of 13 bp separated bya conserved sequence of 8 bp. The site-specific recombination isadvantageously obtained using LoxP sequences or derived sequences, andthe Cre recombinanse.

[0064] The term derived sequence includes the sequences obtained bymodification(s) of the recombination sequences above, conserving thecapacity to specifically recombine in the presence of the appropriaterecombinase. Thus, it may involve reduced fragments of these sequencesor on the contrary fragments extended by addition of other sequences(restriction sites and the like). It may also involve variants obtainedby mutation(s), particularly by point mutation(s).

[0065] According to a preferred embodiment of the invention, thesequences allowing a site-specific recombination are therefore LoxPsequences of the P1 bacteriophage, and the recombination is obtained inthe presence of the Cre protein. In this regard, the recombination maybe obtained by bringing the competent cells directly into contact withthe Cre recombinase, or by expression of the gene encoding the Crerecombinase in the competent cells. Advantageously, the Cre recombinaseis produced in the cell by inducing the expression of the correspondinggene. Thus, the gene encoding the recombinase is advantageously placedunder the control of an inducible promoter, or constructed in aregulatable form. In this regard, there is advantageously used a fusionbetween Cre and the steroid hormone (oestradiol, progesterone and thelike) binding domain allowing the activity of Cre to be regulated andtherefore the recombination event to be induced (Metzger et al., PNAS 92(1995) 6991). More generally, the expression of the recombinase may becontrolled by any strong promoter, regulated or otherwise. Theexpression cassette may be transfected into the competent cells, orintegrated into the genome of the competent cells, as illustrated in theexamples.

[0066] This system therefore makes it possible to generate replicativemolecules producing, in the competent cells, high levels of virus,particularly adenovirus, complementation function. This type ofconstruct is particularly suitable for the complementation of highlydefective genomes, in particular of adenoviral genomes defective for thelate genes. Thus, a specific construct according to the invention isrepresented by a baculovirus comprising, inserted into its genome, atleast a DNA region flanked by two LoxP sequences positioned in directorientation, the said DNA region comprising at least one replicationorigin functional in the competent cells and one nucleic acid encoding acomplementation function of an adenovirus. Advantageously, thecomplementation functions comprise all or some of the immediate-earlygenes present in the E1, E2 and E4 regions. Still more preferably, thecomplementation functions comprise all or some of the immediate-earlygenes and of the delayed-early genes. Preferably, the complementationfunctions allow the complementation of a recombinant adenovirus lackingany coding viral sequence. In particular, the complementation functionsconsist of the whole of the adenoviral genome, with the exception of theITRs and of the packaging region. According to a specific variant, thecomplementation functions consist of a complete adenoviral genomelacking, however, the packaging region (Ad.Psi-). This genome comprisesin particular the ITRs which serve for the replication of the genome inthe competent cells, after excision.

[0067] To bring about the replication of the episomal molecule, thelatter therefore contains a replication origin functional in thecompetent cells used. This replication origin preferably consists of theactual ITR sequences of the adenovirus, which allow substantialamplification of the molecule. It may also be another replication originallowing, preferably, amplification by a factor greater than 20 of theviral DNA in the competent cell. There may be mentioned, by way ofillustration, the origin OriP/EBNA1 of the EBV virus or the E2 region ofthe papilloma virus. It is understood that the ITR sequences of theadenovirus constitute a preferred embodiment.

[0068] For carrying out the process of the invention, the helperbaculovirus(es) are generally used at a Multiplicity of Infection (MOI)allowing a large population of cells to be infected, withoutsignificantly impairing cell viability. Generally, it is moreparticularly between 10 and 1000. The MOI corresponds to the number ofviral particles per cell. The MOI may be easily adjusted by personsskilled in the art depending on the competent cells used, essentially onthe basis of two criteria: the infection efficiency and the possibletoxicity. Advantageously, the MOI used for the helper baculovirus isbetween 20 and 500.

Introduction of the Viral Genome

[0069] As indicated above, the process of the invention comprises theintroduction, into competent cells, of the helper baculovirus and of therecombinant viral genome. In this regard, the genome of the defectiverecombinant adenovirus may be introduced in various ways into thecompetent cell.

[0070] It may, first of all, be a purified defective recombinantadenovirus, advantageously free of RCA. In this case, the competentcells are infected with the defective recombinant adenovirus and withthe helper baculovirus. The infection with the recombinant adenovirusmakes it possible to introduce into the competent cell the correspondinggenome, which is then amplified and encapsidated in order to producestocks at a high titre, free of RCA. This embodiment is particularlyadvantageous for generating first-generation viruses (Ad-ΔE1; Ad-ΔE1,ΔE3). Indeed, these viruses are difficult to produce at high titres,without contamination with RCAs. According to the process of theinvention, it is now possible, starting with a first-generationdefective recombinant adenovirus, by coinfection in a competent cellwith a baculovirus comprising the E1 region, to obtain concentratedstocks, of high quality. This embodiment is also advantageous for theproduction of viruses defective in two or three essential regions oftheir genome (E1, E2, E4 in particular). In general, this embodiment isadvantageous because the efficiency of infection with the adenovirus isvery high (greater than the efficiency of transfection with DNA), andtherefore makes it possible to generate concentrated stocks. In thisembodiment, the recombinant adenovirus and the recombinant baculovirusesare used at multiplicities of infection (MOI) allowing a largepopulation of cells to be infected, without significantly impairing cellviability. The MOI used for the baculovirus is that stated above(between 10 and 1000). As regards the adenoviruses, it is advantageouslybetween 1 and 1000, preferably between 1 and 500, still more preferablybetween 1 and 100. The MOI used for the adenovirus is also adjustedaccording to the cell type chosen. The MOI range may be easilydetermined by persons skilled in the art using, for example, anadenovirus and a baculovirus comprising a separate marker gene, in orderto measure the efficiency of infection and any competition. Morepreferably, the MOI of the adenovirus is less than 50, for examplebetween 1 and 20.

[0071] According to another particularly advantageous embodiment, thegenome of the defective recombinant adenovirus is introduced in the formof DNA. In this case, the genome is introduced by transfection,optionally in the presence of a transfection-facilitating agent (lipids,calcium phosphate and the like). The recombinant genome thus introducedmay be prepared in vitro according to various techniques, and inparticular in E. coli (WO96/25506) or in a yeast (WO95/03400). Thisembodiment is in particular useful for generating a first batch ofdefective recombinant virus, free of RCA, which can then in turn be usedto produce stocks with a high titre according to the precedingembodiment.

[0072] The genome of the defective recombinant adenovirus may also beintroduced using another recombinant baculovirus. According to thisembodiment, the genome of the defective recombinant adenovirus isprepared in vitro, for example as indicated above, and then introducedinto a baculovirus, in the form of a cassette capable of being excisedin the competent cell. According to this embodiment, the competent cellsare put in the presence of a baculovirus carrying the genome of thedefective recombinant adenovirus, and of one or more helperbaculoviruses (carrying the complementation functions). This embodimentis particularly advantageous for the production of highly defectiverecombinant adenoviruses. By virtue of this system, it is indeedpossible to introduce into the population of competent cells highquantities both of the highly defective recombinant genome and of thecorresponding complementation functions.

[0073] In this regard, a process of the invention therefore comprisesthe coinfection of competent cells with a baculovirus carrying thegenome of the defective recombinant adenovirus, and one or more helperbaculoviruses carrying the complementation functions. The MOI valuesused in this embodiment are also between 10 and 1000 for each of thebaculoviruses used.

[0074] Two types of constructs have been prepared in the prior art forthe production of minimum adenoviruses: (1) the transgene(β-galactosidase) cloned between the ITRs, bordered by a uniquerestriction site or (2) the right and left ITRs cloned in directorientation in 5′ of the transgene (Fisher et al., Virology 217 (1996)11; Kumar-Singh et al., Hum. Mol. Genet. 5 (1996) 913). Minimumadenoviruses were produced in the cells 293 by transfection oflinearized (1) or circular (2) DNA, the viral proteins necessary for thereplication and for the encapsidation of the minigenome being providedin trans by a helper virus (AdΔE1). The minimum adenoviruses behave likeinterfering defective (ID) particles and are progressively amplifiedduring successive passages. The major problem posed by the use of thismethodology is the separation of the two types of particles produced,responsible for the contamination of the stocks by the helper virus, andthe very low titres of minimum adenoviruses thus obtained (less than 10⁸pfu/ml).

[0075] The present application makes it possible, for the first time, togenerate minimum adenoviruses using a baculovirus to deliver theadenoviral minigenome and a baculovirus to provide all thetranscomplementation functions (complementing genome).

[0076] The recombinant adenoviral genome is advantageously introducedinto the baculovirus, between two sequences allowing a site-specificrecombination in the competent cells, as described for the helperbaculovirus.

[0077] The present application describes in particular a system for theproduction of a minimum adenovirus using a baculovirus to deliver theadenoviral minigenome with the aid of the loxP/Cre system and abaculovirus to provide all the transcomplementation functions(complementing genome), also with the aid of a Cre/loxP system (seeFIGS. 2-4).

[0078] According to another embodiment, the site-specific recombinationsystem used to deliver the complementation functions is different fromthat used to deliver the genome of the recombinant adenovirus. Inparticular, the LoxP/Cre system may be used to deliver the defectiveadenoviral genome and the AttP/AttB system to deliver thecomplementation function(s).

[0079] The process of the invention thus makes it possible to constructan adenoviral vector deleted of all coding viral sequences andcomprising only the ITRs and the encapsidation signal (minimumadenoviruses). This vector can theoretically accommodate up to 37 kb ofexogenous sequence whereas the cloning capacity of current vectors doesnot exceed 8.5 kb. It thus makes it possible to clone genes of largesize such as the dystrophin gene (14 kb) with all their regulatoryelements (promoter, enhancer, introns and the like) so as to obtain anoptimum expression, in the target tissue. Furthermore, the absence ofany immunogenic viral sequence should increase the duration ofexpression of the transgene in quiescent tissues.

[0080] The genome of AAV or the defective retrovirus may also beintroduced in the form of a virus, a genome or a plasmid, according tothe techniques described above.

Competent Cells

[0081] The process of the invention may be carried out in various typesof cells. For the purposes of the invention, “competent cell” isunderstood to mean a cell permissive to infection by the baculovirus andthe virus to be produced, and allowing a productive viral cycle for thelatter. The capacity to infect cells with these viruses can bedetermined using recombinant viruses expressing a marker gene such asthe E. coli LacZ gene. It is preferably a mammalian cell, still morepreferably a cell of human origin. The competent cells used may bequiescent cells or actively dividing cells, established lines or primarycultures. They are advantageously mammalian cells compatible with anindustrial use, that is to say without a known pathogenic character,capable of being cultured and, where appropriate, of being stored underappropriate conditions. Advantageously, the cells used are hepatic,muscular, fibroblastic, embryonic, nerve, epithelial (pulmonary) orocular (retinal) cells. There may be mentioned, by way of nonlimitingexample, the cells 293 or any derived cell comprising an additionalcomplementation function (293E4, 293E2a, and the like), the A549 cells,the HuH7 cells, the Hep3B cells, the HepG2 cells, the humanretinoblastic cells (HER, 911), the HeLa cells, the 3T3 cells or the KBcells.

[0082] To carry out the process of the invention, the genome of therecombinant virus and the baculovirus may be introduced into thepopulation of competent cells simultaneously or spaced out over time.Advantageously, the cells are brought into contact both with therecombinant genome and the helper baculovirus. In the case of a systemgenerating replicative molecules in vivo, the recombinase is introducedor expressed beforehand, simultaneously or subsequently.

[0083] The production of the viruses generally leads to the lysis of thecells. The viruses produced can therefore be harvested after cell lysis,according to known purification methods. They can then be packaged invarious ways depending on the desired use. Moreover, to avoid any riskof contamination of the viral stock with possible traces ofbaculoviruses that have not penetrated into the competent cells (helperbaculovirus or baculovirus providing the recombinant viral genome), itis possible to apply the following techniques:

[0084] it is possible to purify the adenoviruses by chromatographyaccording to the method described in application FR96/08164. Thistechnique makes it possible to separate the adenovirus from any possibleresidual baculovirus;

[0085] it is also possible to cause organic solvents (for example ether,chloroform) to act on the stocks of purified adenovirus. Indeed, thebaculovirus is an enveloped virus (glycoprotein envelope), and istherefore very sensitive to any organic solvent (which extracts thelipids from its envelope); in contrast, the adenovirus is not enveloped,and the same solvents have no effect on it;

[0086] it is also possible, by CsCl gradient purification, to separate,by density, any residual baculovirus and the recombinant virus.

[0087] These three methods can be used independently or conjointly.Moreover, any other method known to a person skilled in the art can alsobe used.

Use of the Viruses

[0088] The viruses thus produced can be used for the cloning, transferand expression of genes in vitro, ex vivo or in vivo. Such genes ofinterest are, for example, genes encoding enzymes, blood derivatives,hormones, lymphokines: interleukins, interferons, TNF, and the like (FR9203120), growth factors, neurotransmitters or their precursors ofsynthesis enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF,bFGF, NT3, NT5, and the like, apolipo proteins: ApoAI, ApoAIV, ApoE, andthe like (WO94/25073), dystrophin or a minidystrophin (WO93/06223),tumour suppressor genes: p53, Rb, Rap1A, DCC, k-rev, and the like(WO94/24297), genes encoding factors involved in clotting: Factors VII,VIII, IX and the like, suicide genes: thymidine kinase, cytosindeaminase and the like, or all or part of a natural or artificialimmunoglobulin (Fab, ScFv, and the like, WO94/29446), and the like. Thegene of interest may also be a gene or an antisense sequence, whoseexpression in the target cell makes it possible to control theexpression of genes or the transcription of cellular mRNAs. Suchsequences may, for example, be transcribed, in the target cell, intoRNAs complementary to cellular mRNAs and thus block their translationinto protein, according to the technique described in Patent EP 140 308.The gene of interest may also be a gene encoding an antigenic peptide,capable of generating an immune response, for the production ofvaccines. It may be in particular antigenic peptides specific for theEpstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573),the pseudorabies virus, or specific for tumours (EP 259 212). The genemay be any DNA (gDNA, cDNA and the like) encoding a product of interest,potentially including the appropriate expression signals (promoter,terminator and the like).

[0089] These viruses may be used in vitro for the production of theserecombinant proteins. They may also be used, still in vitro, to studythe mechanism of action of these proteins or to study the regulation ofthe expression of genes or the activity of promoters.

[0090] They may also be used in vivo, for the creation of animal modelsor of transgenic animals. They may also be used for the transfer andexpression of genes in vivo, in animals or man, in gene or cell therapyprocedures.

[0091] The present application will be described in greater detail withthe aid of the following examples which should be considered asillustrative and nonlimiting.

Legend to the Figures

[0092]FIG. 1: Schematic representation of the production of athird-generation defective recombinant adenovirus (defective for the E1and E4 functions) using a baculo-E1, E4.

[0093] FIGS. 2-4: Schematic representation of the production of aminimum adenovirus using a first baculovirus to introduce the defectiveadenoviral genome and a second baculovirus to introduce thecompleentation functions.

[0094]FIG. 5: Schematic representation of the production of arecombinant AAV defective for the Rep and Cap functions using abaculo-Rep/Cap.

EXAMPLES

[0095] 1. Cells Used

[0096] The cells used within the framework of the invention may beobtained from any cell line or population capable of being infected byan adenovirus or an AAV or a retrovirus or by a baculovirus, compatiblewith a use for therapeutic purposes. It is more preferably a mammalian,particularly human, cell. There may be mentioned more particularly:

The Cells of the 293 Line:

[0097] The 293 line is a human embryonic kidney cell line containing theleft end (about 11-12%) of the genome of the serotype 5 adenovirus(Ad5), comprising the left ITR, the encapsidation region, the E1 region,including E1a, E1b, the region encoding the pIX protein and part of theregion encoding the pIVa2 protein (Graham et al., J. Gen. Virol. 36(1977) 59). This line is capable of transcomplementing recombinantadenoviruses defective for the E1 region, that is to say lacking all orpart of the E1 region, and of producing viral stocks having high titres.

The Cells of the A549 Line

[0098] Cells complementing the E1 region of the adenovirus wereconstructed from A549 cells (Imler et al., Gene Ther. (1966) 75). Thesecells contain a restricted fragment of the E1 region, lacking the leftITR, placed under the control of an inducible promoter.

The Cells of the HER Line

[0099] The human embryonic retinal (HER) cells can be infected with anadenovirus (Byrd et al., Oncogene 2 (1988) 477). Adenovirusencapsidation cells prepared from these cells have been described forexample in application WO94/28152 or in the article by Fallaux et al.(Hum. Gene Ther. (1996) 215). There may be mentioned more particularlythe 911 line comprising the E1 region of the genome of the Ad5adenovirus, from nucleotide 79 to nucleotide 5789, integrated into thegenome of HER cells. This cell line allows the production of virusesdefective for the E1 region.

The IGRP2 Cells

[0100] The IGRP2 cells are cells obtained from cells 293, by integrationof a functional unit of the E4 region under the control of an induciblepromoter. These cells allow the production of viruses defective for theE1 and E4 regions (Yeh et al., J. Virol (1966) 70).

The VK Cells

[0101] The VK cells (VK2-20 and VK10-9) are cells obtained from cells293, by integration of the entire E4 region under the control of aninducible promoter, and the region encoding the protein pIX. These cellsallow the production of viruses defective for the E1 and E4 regions(Krougliak et al., Hum. Gene Ther. 6 (1995) 1575).

The 293E4 Cells

[0102] The 293E4 cells are cells obtained from cells 293, by integrationof the entire E4 region. These cells allow the production of virusesdefective for the E1 and E4 regions (WO95/02697; Cancer Gene Ther.(1995) 322).

[0103] The Sf9 and Sf21 cells are embryonic Lepidoptera cells. Thesecells are accessible in collections (No. CRL-1711 ATCC) as well as theirculture conditions. They are also commercially available (Gibco). Seealso King and Possee: The baculovirus expression system, London: Chapmanand Hall, 1992.

The Human Hepatocytic Cells

[0104] The HepG2 and Hep3B and HuH7 cells are human lines derived fromhepatocarcinomas. They are accessible in depository collections andtheir properties have been described for example in Patents U.S. Pat.No. 4,393,133 and 4,393,133.

[0105] Human cell line KB: Derived from a human epidermal carcinoma,this line is accessible at the ATCC (ref. CCL17) as well as theconditions allowing its culture.

[0106] Human cell line Hela: derived from a carcinoma of the humanepithelium, this line is accessible at the ATCC (ref. CCL2) as well asthe conditions allowing its culture.

[0107] Cell line W162: These cells are Vero cells comprising, integratedinto their genome, the E4 region of the Ad2 adenovirus. These cells havebeen described by Weinberg et al., (PNAS 80 (1983) 5383).

[0108] 2. Infection of Human Cells with a Recombinant Baculovirus

[0109] This example describes the capacity of baculoviruses to infectcells of human origin.

[0110] Human cells (particularly 293 or derivatives thereof) areinfected with various dilutions of a solution of recombinant baculovirusexpressing the LacZ gene under the control of the RSV LTR. 48 hoursafter infection, the appearance of blue cells is revealed, demonstratingthe infectability of these cells by a baculovirus.

[0111] 3. Construction of Baculoviruses Expressing the E1 Region of theAdenovirus and of a Corresponding Defective Adenovirus

[0112] 3-1 Cloning of Two Cassettes for the Expression of E1

[0113] The plasmid AE2 is obtained from the cloning, in PCRII(Invitrogen) of the product of the PCR performed on pBRE1 with theoligonucleotides 5′-TCCTTGCATTTGGGTAACAG-3′ and5,′-GCGGCCGCTCAATCTGTATCTTC-3′; this PCR product contains nucleotides3198 to 3511 of Ad5, that is to say the 3′ end of the E1B region. Theplasmid pBRE1 contains nucleotides 1 to 5788 of Ad5 cloned into pBR322,deleted roughly of nucleotides 1300 to 2300.

[0114] The plasmid AE3 is derived from the cloning of the NotI-KpnIfragment of AE2, containing the PCR product, into pCDNA3 (Invitrogen)digested with NotI-KpnI. It contains nucleotides 3198 to 3511 of Ad5followed by the polyadenylation site of BGH.

[0115] The plasmid AE4 is derived from the cloning of the BglII-PvuIIfragment of AE3 into pBRE1. AE4 is a plasmid containing the following E1expression cassette:

[0116] nucleotides 1 to 3511 of Ad5, that is to say left ITR,encapsidation sequences, E1A promoter, E1A gene, E1B promoter, E1B gene

[0117] the polyA of the bovine growth hormone (BGH) obtained frompCDNA3.

[0118] pBRE1 was digested with BstNI, and then digested with T4 DNApolymerase in order to fill the protruding 5′ end, and then digestedwith XbaI. The fragment thus generated containing nucleotides 391 to1339 of Ad5 was introduced into pic20H digested with SmaI-XbaI(nonmethylated site), to give the plasmid AE5.

[0119] The plasmid AE6 is derived from the cloning of the EcoRI-SmaIfragment of AE5 into AE4 digested with EcoRI-SmaI. AE6 is a plasmidcontaining the following E1 expression cassette:

[0120] nucleotides 391 to 3511 of Ad5, that is to say “reduced” promoterof E1A, E1A gene, E1B promoter, E1B gene,

[0121] the polyA of the bovine growth hormone (BGH) obtained frompCDNA3.

[0122] 3-2 Cloning of these Two E1 Cassettes into a Baculovirus

[0123] The EcoRI-SphI fragments (SphI protruding 5′ end which has beenmade blunt beforehand by digestion with T4 DNA polymerase) of theplasmids AE4 and AE6 are cloned into the plasmid pAcSG2 (Pharmingen)between the EcoRI and EcoRV sites. This generates the plasmids AE14 andAE15 respectively. In both cases, the E1 region is introduced into thelocus of the polyhedrin gene (polyhedrin gene+polyhedrin promoter beingdeleted).

[0124] The plasmids AE14 and 15 are cotransfected with the DNA of thebaculovirus BaculoGold derived from the AcNPV strain (Pharmingen) intoSf9 insect cells, in order to generate the two corresponding recombinantbaculoviruses BacAE14 and BacAE15, carrying the two cassettes for theexpression of E1 integrated into the locus of the polyhedrin gene.

[0125] 3-3 Cloning of a recombinant Adenovirus Carrying a New E1Deletion

[0126] The plasmid pCO1 (WO96/10088) was digested with BstXI, and thendigested with T4 DNA polymerase in order to remove the protruding 3′end, and then digested with RsaI. The fragment thus generated containingnucleotides 3513 to 4607 of Ad5 was introduced into pBS-SX+ (Stratagene)linearized with EcoRV and then digested with calf alkaline phosphatase,to generate the plasmid AE0.

[0127] The plasmid pMA37 is obtained by ligation of the fragments:

[0128] EcoRV-NsiI of pXL2756, containing sacB. pXL2756 possesses thecounter-selection gene SacB free of its EcoRI and KpnI sites, thekanamycin resistance gene, a multiple cloning site and a replicationorigin ColE1,

[0129] NdeI-NsiI of pCO1 (containing the adeno sequences)

[0130] SalI (protruding 5′ end filled with T4 DNA polymerase)-AseI ofpXL2756 (kanamycin-resistant vector).

[0131] pMA37 therefore contains:

[0132] the kanamycin resistance gene

[0133] the SacB gene conferring sucrose sensitivity on bacteriaexpressing it

[0134] sequences 1 to 382 (HinfI) of Ad5 followed by sequences 3446 to4415 (NsiI) of Ad5; there is no transgene.

[0135] The plasmid AE11 was constructed by introducing the XhoI-NsiIfragment of AE0 into pMA37 digested with SalI-NSiI. It thus contains:

[0136] the kanamycin resistance gene

[0137] the SacB gene conferring sucrose sensitivity on bacteriaexpressing it,

[0138] sequences 1 to 382 (HinfI) of Ad5 followed by sequences 3513 to4415 (NsiI) of Ad5; there is no transgene.

[0139] From the plasmid AE11 are then constructed the suicide shuttlevectors (by insertion of the transgene of interest) allowing theconstruction of recombinant adenoviruses by recombination in E. Coli.AE11 contains no sequence homologous with the plasmid AE6. The plasmidsAE11 and AE4 have in common sequences 1 to 382 (HinfI) of Ad5 upstreamof E1A, but there is no homology downstream of the E1 cassette betweenthese two plasmids. Thus, there can be no generation of RCA byhomologous recombination between an adeno carrying the E1 deletionexisting in AE11 (that is to say 382-3513) and the baculoviruses BacAE14or BacAE15.

[0140] 3-4 Construction of a First-generation Recombinant Adenovirus

[0141] In the first instance, a stock of BacAE14 or 15 is preparedaccording to conventional techniques. Next, the competent cells (forexample HuH7) are transfected with 5 μg of plasmid pXL2822 digested withPacI (the plasmid pXL2822 contains all the Ad5 deleted for E1 (382-3446or 382-3513) and E3 (28592-30470) and carries a cassette CMV-βGal), andinfected, simultaneously or otherwise, at an MOI between 10 and 1000,with BacAE14 or 15. When the cells are lysed, the transfectionsupernatant is harvested, and then applied onto “fresh” competent cellspreviously or simultaneously infected with BacAE14 or 15 (MOI 10 to1000), so as to amplify the adenovirus Ad2822, and so on until a stockof Ad2822 is obtained. The monitoring of the amplification of Ad2822 isfacilitated by the presence of lacZ in this virus. On eachamplification, the supernatant is thus pseudotitrated on W162. Thegenome of the virus is analysed during amplifications so as to check itsintegrity. Finally, this strategy has the advantage of not generatingRCA in the adenovirus stocks thus produced. This absence ofcontamination is also verified.

[0142] 4. Construction of a Baculovirus Expressing the E1 and E4 Regionsof the Adenovirus

[0143] 4-1 Construction of the Baculovirus E1,E4 (Bac.E1-E4)

[0144] The protocol used is the following: the E1 and E4 regions arecloned in reverse orientation to the locus of the polyhedrin (Ph) gene,into the shuttle vector pBacPAK8 (Clontech, USA) to give pBacE1-E4. Therecombinant baculovirus is then isolated according to conventionaltechniques by cotransfection of pBacE1-E4 and of the DNA of thebaculovirus BacPAK6 into Sf9 cells (Kitts and Possee, Biotechniques14(5) (1993) 810). The presence of E1 and E4 in the genome of therecombinant baculoviruses is then checked by PCR using infected Sf9cells. The transcription of E1 and E4 in the Huh7 cells infected withthe purified recombinant baculovirus is analysed by RT-PCR usingcytoplasmic RNAs.

[0145] For the construction of the E1-E4 baculovirus, various fragmentscarrying E1 may be introduced. The fragments used in this example arethose described in Example 3 for the construction of the Baculovirus-E1,containing the E1 regions under the control of their own promoter(reduced E1a promoter and E1b promoter). The E4 fragments used are thefollowing:

[0146] fragment MaeII-MscI 32720-35835 carrying the entire E4

[0147] fragment BglII-PvuII 34115-33126 carrying the frame ORF6

[0148] fragment BglII-BglII 34115-32490 carrying the frames ORF6-ORF6/7

[0149] fragment PvuII-AluI 34801-334329 carrying the frame ORF3

[0150] These fragments are placed alternatively under the control of theE4 promoter or of different promoters, particularly of the HSV-TK or CMVpromoter or of the RSV-LTR.

[0151] The positions given above refer to the sequence of the wild-typeAd5 adenovirus as published and accessible on database. Although someminor variations may exist between the various adenovirus serotypes,these positions are generally transposable to other serotypes, and inparticular to Ad2, Ad7, Ad12 and Ad CAV-2.

[0152] 4-2 Transcomplementation of AdΔE1ΔE4-LacZ with Bac.E1-E4

[0153] The production of the adenovirus AdΔE1ΔE4-LacZ is obtained byintroduction into the competent cells of the E1, E4 baculovirus preparedin Example 4-1 and of the adenoviral genome defective for E1 and E4 (seeFIG. 1). The optimum conditions for the production of AdΔE1ΔE4-LacZ inthe competent cells, by transcomplementation with the purifiedBac.E1-E4, are analysed. The AdΔE1ΔE4 titre is determined as number ofβ-galactosidase transduction units (t.d.u.) in the W162 line and thetranscomplementation efficiency obtained is compared with that of theencapsidation line IGRP2.

[0154] 5. Construction of a Baculovirus Complementing the Whole of theAdenovirus Genome

[0155] Although the simultaneous expression of several proteins, whichmay represent up to 13 kb of sequence, from a single virus has beenreported (Belyaev and Roy, NAR 21 (1993) 1219), the maximum cloningcapacity of the baculovirus is not highly documented. This example nowshows that it is possible to clone the adenovirus genome deleted of itsencapsidation signal (Ad.Psi-) and bordered by two loxP sites[loxP-ITR-ITR to E4 -loxP] into the baculovirus. The infection ofcompetent cells expressing the recombinase Cre, in an inducible manneror otherwise (Cre or Cre-ER line, see Example 7), with this recombinantbaculovirus and the activation of the recombinase Cre, allow theexcision and the circularization of the adenoviral genome. The latter isthen capable of transducing the early genes, of replicating and ofactivating the late genes, but incapable of being encapsidated, and thusserves as helper for the production of a minimum adenovirus (see FIGS.2-4). In this system, the production of the minimum adenoviruses isbased on the co-infection by two baculoviruses and the realization of 2recombination events between the loxP sites. This approach has theadvantage of not generating adenoviral particles from the helper virus.

[0156] The construction of the Ad.Psi-genome is carried out in E. coli.For that, the complete genome of the Ad5 adenovirus is cloned into aprokaryotic cloning vector, ITRs attached. The Psi sequence is deletedby enzymatic cleavage and ligation, or by site-directed mutagenesis. ALoxP sequence is then introduced on either side of the adenoviralgenome, in parallel orientation. The resulting construct [loxP-ITR-ITR,ΔPsi to E4-loxp] is then cloned into a shuttle vector allowingrecombination with a baculovirus, according to the strategy described inExample 3. The recombinant baculovirus obtained, called BacAd.Psi-, isthen isolated according to conventional methods.

[0157] 6. Construction of a Baculovirus Comprising the Genome of theDefective Recombinant Adenovirus in Excisable Form

[0158] This example describes the construction of a baculovirus whichmakes it possible to provide in the competent cells the genome of thedefective recombinant adenovirus. More particularly, the recombinantadenovirus is defective for the whole of the coding regions, and retainsonly the ITR and Psi regions (minimum adenovirus, or AdΔ).

[0159] 6-1 Construction of a Minigenome (AdΔ) in E. Coli

[0160] A plasmid p[loxP-(ITR-ITR-Psi-P.CMV-LacZ-pA)-loxP] isconstructed. For that, a copy of the ITR sequence of the adenovirus isisolated by enzymatic cleavage and/or amplified by PCR, and then clonedupstream of the ITR-Psi sequence contained in the shuttle vector of theadenovirus pGY63. This vector is derived from pCO1 (WO96/10088) andpossesses the LacZ gene under the control of the immediate-earlypromoter of the cytomegalovirus (P.CMV) ending with the polyadenylationsignal of the SV40 virus (pA), cloned between the ITR-Psi sequence andthe gene encoding pIX. The region (ITR-ITR-Psi-P.CMV-LacZ-pA) of thisvector (corresponding to a minimum adenovirus genome) is then isolatedby enzymatic cleavage and cloned between the LoxP sites into themultiple cloning site of the plasmid pBS246 (Gibco), to generate theplasmid p[loxP-(ITR-ITR-Psi-P.CMV-LacZ-pA)-loxP]. The capacity toproduce adEnovirus minigenomes from a circular DNA and to encapsidatethem is then tested by transfection of this plasmid into the IGRP2 lineinfected with Ad.ΔE1ΔE4 expressing the recombinase Cre (AdCre). Theminimum adenoviruses are amplified by a few successive passages of thesupernatant from the transfection in the IGRP2 line. They are thenpurified by isopycnic caesium chloride gradient centrifugation andquantified by pseudotitration in the W162 line. It is understood thatthe LacZ gene can be easily replaced by any other nucleic acid ofinterest, by conventional molecular biology techniques.

[0161] 6.2 Cloning of an Excisable Minigenome into a Baculovirus

[0162] The construct carrying the minigenome AdΔ bordered by the twoloxP sites described above is cloned at the P10 locus of the baculovirusinto the shuttle vector pAcUW1 (Pharmingen, USA). The baculovirusBac.AdΔ is then produced and isolated by conventional techniques ofcotransfection into the abovementioned Sf9 cells, and selected by itsphage phenotype (white), after staining with X-Gal. This baculovirustherefore carries a highly defective adenoviral genome, flanked by twoloxP regions in direct orientation.

[0163] 6-3 Production of AdΔ by Transcouplementation with theBaculovirus BacAdPsi-

[0164] Competent cells are simultaneously co-infected with thebaculovirus BacAdPsi- (described in Example 5), carrying thetranscomplementation functions of the whole of the adenoviral genome,and with the baculovirus Bac.AdΔ carrying the genome of thePseudoAdenovirus (described above). The recombinase Cre is providedeither by adding the protein into the culture medium, or by transfectingthe cells with a plasmid or a virus (baculovirus) expressing Cre, or byexpression of a cassettee stably integrated into the genome of the line(as described in Example 7). The minimum adenovirus is amplified bysuccessive passages of the culture supernatants of cells co-infectedwith BacAd.Psi- and with the supernatant, and then purified and titratedaccording to the techniques mentioned above. This technique makes itpossible to obtain AdΔ as sole virus, which allows its isolation and itspurification by conventional techniques. In addition, the titresobtained are compatible with an industrial use.

[0165] 7. Construction of a Line Expressing the Cre Protein

[0166] A line expressing Cre, in an inducible manner or otherwise, isconstructed in order to increase the efficiency of recombination betweenthe loxP sites in the baculovirus of the invention (for example Bac.AdΔand BacAd.Psi-) and to control the expression of Cre. In this construct,Cre is expressed alone or in the form of a C-terminal fusion proteinwith the oestradiol receptor (ER) binding domain (Metzger et al., 1996,cited above), under the control of a ubiquitous promoter, preferably astrong promoter inducible or otherwise. More particularly, the promotersused are the pGRE5 promoter, the metallothionin promoter, the SV40promoter or the promoter of the HSV-TK gene.

[0167] To construct these Cre lines, the compEtent cells arecotransfected with two plasmids, one containing the Cre expressioncassette (Cre or Cre-ER) and the other that for a selectable marker(Neo). G418-resistant clones are selected, the Cre activity in theseclones is tested by transfection of the plasmidp(P.CMV-loxP-ATG-stop-pA-LoxP-LacZ). This plasmid contains the LacZ geneinactivated by introducing between the promoter (P.CMV) and thebeginning of LacZ a succession of stop codons in the three readingframes and the signal for termination of transcription and thepolyadenylation signal of the SV40 virus, bordered by two loxP sites.The expression of Cre in the clones, in the presence or otherwise of theinducer (oestradiol), is then revealed by the β-galactosidase activityinduced by recombination between the two loxP sites. Several clonesstably expressing the fusion protein Cre-ER or the protein Cre alonefrom the promoters and the competent cells specified below are thusselected. These clones can be used for the production of virusesaccording to the invention. HSV-TK SV40 MMTV GRE5 Competent promoterpromoter promoter promoter cell Cre- Cre- Cre- Cre- Recombinase Cre ERCre ER Cre ER Cre ER 293 #1  #7 #13 #19 #25 #31 #37 #43 IGRP2 #2  #8 #14#20 #26 #32 #38 #44 Huf7 #3  #9 #15 #21 #27 #33 #39 #45 HepG2 #4 #10 #16#22 #28 #34 #40 #46 HER #5 #11 #17 #23 #29 #35 #41 #47 Vero #6 #12 #18#24 #30 #36 #42 #48

1. Process for the production of defective recombinant viruses accordingto which the genome of the defective recombinant virus and a baculoviruscomprising all or some of the functions necessary for thetranscomplementation of the defective recombinant genome are introducedinto a population of competent cells.
 2. Process according to claim 1,characterized in that the baculovirus comprises all the functionsnecessary for the transcomplementation of the defective recombinantgenome.
 3. Process according to claim 1, characterized in that thebaculovirus comprises some of the functions necessary for thetranscomplementation of the defective recombinant genome, the rest beingprovided by the competent cell.
 4. Process according to claim 1,characterized in that the functions necessary for thetranscomplementation of the defective recombinant genome are provided byseveral baculoviruses.
 5. Process according to claim 1, characterized inthat the defective recombinant virus is a defective recombinantadenovirus.
 6. Process according to claim 5, characterized in that thegenome of the recombinant adenovirus is defective for one or morefunctions chosen from E1, E2, E3, E4, L1-L5, pIX and IVa2 and thebaculovirus comprises all the functions necessary for thetranscomplementation of the defective recombinant genome.
 7. Processaccording to claim 5, characterized in that the genome of therecombinant adenovirus is defective for one or more functions chosenfrom E1, E2, E3, E4, L1-L5, pIX and IVa2, the baculovirus comprises someof the functions necessary for the transcomplementation of the defectiverecombinant genome, the rest of the functions being provided by one ormore other baculoviruses and/or by the competent cell.
 8. Processaccording to claim 5, characterized in that the helper baculoviruscomprises all or part of the E1 region of the adenovirus, allowing thecomplementation of a recombinant adenovirus genome defective for the E1region.
 9. Process according to claim 5, characterized in that thehelper baculovirus comprises all or part of the E2 region of theadenovirus, allowing the complementation of a recombinant adenovirusgenome defective for the E2 region.
 10. Process according to claim 5,characterized in that the helper baculovirus comprises all or part ofthe E4 region of the adenovirus, allowing the complementation of arecombinant adenovirus genome defective for the E4 region.
 11. Processaccording to claim 5, characterized in that the helper baculoviruscomprises all or part of the E1 and E4 regions of the adenovirus,allowing the complementation of a recombinant adenovirus genomedefective for the E1 and E4 regions.
 12. Process according to claim 5,characterized in that the recombinant adenovirus genome lacks any codingviral region and the helper baculovirus comprises all the functionsallowing its complementation.
 13. Process according to claim 12,characterized in that the baculovirus comprises the whole of anadenoviral genome, with the exception of the encapsidation region andpossibly of the ITRs.
 14. Process according to claim 1, characterized inthat the complementation functions present in the baculovirus and thegenome of the defective recombinant virus do not comprise a zone ofhomology capable of giving rise to recombination.
 15. Process accordingto claim 14, characterized in that a recombinant adenovirus genomedefective for the E1 and possibly E3 region is introduced into thecompetent cells, these cells are infected, simultaneously or otherwise,with a baculovirus comprising the E1 region, the adenovirus E1 regionpresent in the baculovirus and the genome of the defective recombinantadenovirus comprising no zone of homology capable of giving rise torecombination.
 16. Process according to claim 15, characterized in thatthe baculovirus comprises a fragment 391-3511 of the Ad5 adenovirus andin that the genome of the recombinant adenovirus defective for the E1region carries a larger deletion.
 17. Process according to claim 16,characterized in that the baculovirus comprises a fragment 391-3511 ofthe Ad5 adenovirus and in that the genome of the recombinant adenovirusdefective for the E1 region carries a deletion covering nucleotides383-3512 inclusive.
 18. Recombinant baculovirus comprising, insertedinto its genome, a nucleic acid encoding a complementation function of adefective virus placed under the control of a heterologous promoter. 19.Baculovirus according to claim 18, characterized in that thecomplementation function is chosen from all or some of the functionsencoded by the E1, E2, E4, L1-L5, pIX and IVa2 regions of theadenovirus, alone or in combinations.
 20. Baculovirus according to claim18, characterized in that the complementation function is chosen fromall or some of the functions encoded by the Rep and Cap regions of theAAV, alone or in combinations.
 21. Baculovirus according to claim 18,characterized in that the complementation function is chosen from all orsome of the functions encoded by the gag, pol and env regions of aretrovirus, alone or in combinations.
 22. Baculovirus according to claim18, characterized in that the nucleic acid encoding the complementationfunction consists of a DNA corresponding to a fragment of a genome ofthe virus comprising the corresponding region.
 23. Baculovirus accordingto claim 22, characterized in that the nucleic acid encoding thecomplementation function consists of a DNA corresponding to a fragmentof a genome of serotype Ad2 or Ad5 adenovirus.
 24. Baculovirus accordingto claim 18, characterized in that the promoter consists of the promoterregion naturally responsible for the expression of the complementationfunctions.
 25. Baculovirus according to claim 18, characterized in thatthe promoter is a strong cellular or viral promoter, regulated orotherwise.
 26. Baculovirus according to claim 19, characterized in thatthe complementation function comprises the E1 region of an adenoviralgenome or only a part thereof comprising at least the E1a region. 27.Baculovirus according to claim 19, characterized in that thecomplementation function comprises the E4 region of an adenoviral genomeor only a part thereof comprising at least the frame ORF3 or ORF6. 28.Baculovirus according to claim 19, characterized in that it comprisesall the coding regions of an adenoviral genome.
 29. Baculovirusaccording to claim 28, characterized in that it comprises a completeadenoviral genome, lacking the encapsidation region.
 30. Baculovirusaccording to claim 18, characterized in that it is an AcNPV strain. 31.Baculovirus according to claim 18, characterized in that the nucleicacid is introduced at the level of the polyhedrin locus or of the p10locus.
 32. Baculovirus according to claim 18, characterized in that thenucleic acid is introduced in the form of a cassette which is capable ofbeing excised in the competent cell.
 33. Recombinant baculoviruscomprising, inserted into its genome, at least one DNA region flanked bytwo sequences allowing a site-specific recombination and positioned indirect orientation, the said DNA region comprising at least onereplications origin functional in competent cells and a nucleic acidencoding a complementation function of a virus.
 34. Baculovirusaccording to claim 33, characterized in that the sequences allowing asite-specific recombination are LoxP sequences of the P1 bacteriophage,and the recombination is obtained in the presence of the Cre protein.35. Process according to claim 5, characterized in that the defectiverecombinant genome is introduced into the cell by infection with anadenovirus comprising the said genome.
 36. Process according to claim 5,characterized in that the defective recombinant genome is introducedinto the cell by transfection.
 37. Process according to claim 1,characterized in that the defective recombinant genome is introducedinto the cell with a recombinant baculovirus, distinct from thebaculovirus carrying the complementation functions.
 38. Recombinantbaculovirus comprising, inserted into its genome, at least one DNAregion flanked by two sequences allowing a site-specific recombinationand positioned in direct orientation, the said DNA region comprising atleast one replication origin functional in competent cells and adefective adenovirus genome.
 39. Baculovirus according to claim 38,characterized in that the defective recombinant adenovirus genomecomprises essentially the ITR regions, the encapsidation sequence and anucleic acid of interest.
 40. Process for the production of defectiverecombinant adenoviruses, characterized in that a population ofcompetent cells is infected with a baculovirus according to claim 33 andwith a baculovirus according to claim 38, the cells are put in thepresence of the recombinase allowing the site-specific recombination,and then the adenoviruses produced are recovered.
 41. Process accordingto claim 1, characterized in that the population of competent cells is apopulation of hepatic, muscle, fibroblast, embryonic, epithelial(particularly pulmonary), ocular (particularly retinal) or nerve cells.42. Process according to claim 41, characterized in that the populationof competent cells is chosen from the cells 293 or any derived cellcomprising an additional complementation function, A549, HuH7, Hep3B,HepG2, HER, 911, HeLa or KB.
 43. Purified viral preparation obtainedusing the process according to claim 1.